Fig 5. Response of CCND1 3’UTR and protein expression towards miR-17 family.

(A) The CCND1 3’UTR was cloned behind the luciferase reporter gene of the pMIR vector and the potential binding site for the indicated miRNAs in the 3’UTR was additionally mutated by site directed mutagenesis (CCND1 mut). The reporter gene construct was expressed with the miRNA expression construct or with the empty pSG5 vector as control in the indicated combinations. Results represent the mean of at least four independent experiments performed in duplicates. The dashed line represents the luciferase activity of the empty luciferase reporter plasmid with the empty pSG5 vector which was set to 100% (***, p<0.001). (B) LNCaP cells were transfected either with control vector or miRNA expression vectors. 48 hours post-transfection the protein expression of CCND1 was determined by Western blot using ß-actin as loading control. The densitometrical quantification of Western Blots represents the relative downregulation of CCND1 expression as determined in three independent experiments in relation to the corresponding ß-actin band as loading control.